Polypeptide and hyaluronic acid coatings

ABSTRACT

A polyelectrolyte coating comprises at least one polycationic layer consisting of at least one polyarginine as herein defined and at least one polyanionic layer consisting of hyaluronic acid. The polyelectrolyte coating has a biocidal activity and the invention thus further refers to the use of said polyelectrolyte coating for producing a device, in particular a bacteriostatic medical device, more particularly an implantable device, comprising said polyelectrolyte coating, and a method for preparing said device and a kit. Also disclosed is a method of preventing a bacterial infection in an individual.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation-in-part of PCT/EP2017/060378 filedMay 2, 2017, herein incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a polyelectrolyte coating comprising atleast one polycationic layer consisting of at least one polyarginine asherein defined and at least one polyanionic layer consisting ofhyaluronic acid. The polyelectrolyte coating has a biocidal activity andthe invention thus further refers to the use of said polyelectrolytecoating for producing a device, in particular a bacteriostatic medicaldevice, more particularly an implantable device, comprising saidpolyelectrolyte coating, and a method for preparing said device and akit. The invention further refers to a method of preventing a bacterialinfection in an individual.

BACKGROUND OF THE INVENTION

Nosocomial infections (also called health care associated infections),the fourth leading cause of disease in industrialized countries, are amajor health issue. Year by year, implantation of prostheses and medicaldevices is increasing. In the meantime the prevalence of nosocomialinfections related to implants, which are reported in the literature, isconstantly on the rise. It is known that half of all nosocomialinfections worldwide involve a medical device. Accordingly, the mostsignificant hospital-acquired infections, based on frequency andpotential severity, are those related to procedures e.g. surgical siteinfections and medical devices, including urinary tract infection incatheterized patients, pneumonia in patients intubated on a ventilatorand bacteremia related to intravascular catheter use.

Some key factors for the increase of medical-device related infectionsare i) ageing of the population, ii) multiple drug resistance bacteria,iii) poor development of new designed antibiotic molecules. In the caseof medical devices like implants, the surgical site is an attractivetarget for pathogens and leads to early complications. To prevent suchinfections associated with implants, a local treatment for the first 6hours post-implantation is of particular interest.

Innovative, bioactive, smart coatings and materials for reducingnosocomial infections are urgently needed to slow this trend.

The alternate deposition of polycations and polyanions on a substrateleads usually to the formation of a coating, called polyelectrolytecoating. Those polyelectrolyte coatings consist of a multilayeredstructure and their thickness increases with the number of depositionsteps. The potential applications of these kinds of coatings arewidespread ranging from energy storage devices to anti-fogging coatingsand bioactive substrates. In this latter area, antimicrobial coatingsare receiving extensive attention due to their importance in the fightagainst nosocomial infections.

One strategy for antimicrobial coatings consists in the design ofanti-adhesive coatings to inhibit attachment and growth of pathogens onthe device. These anti-adhesive coatings thus prevent biofilm formation.However, as the growth of the pathogen is not inhibited by this method,the risk of colonization of another surrounding site is high, inparticular in the case of fragile and immunodeficient people.

Another strategy consists in the design of bactericidal coatings. Thesecoatings usually release antimicrobial agents such as antibiotics thatwere incorporated in the coating structure either during its buildup orby diffusion in the coating after buildup. The release of the activecompound can be triggered for example by enzymatic degradation of thecoating or by a pH change. It can also take place naturally due tohydrolysis of one of the film constituent.

These are interesting approaches; however the release profile in situ ofbactericidal coatings should be perfectly controlled to avoid negativeeffects of overdosed delivered drugs. Moreover, in most of the cases,the release is passive which means antimicrobial agents are delivered inthe presence or absence of bacteria. To circumvent these drawbacks,contact-killing strategies could be more advantageous and consist indamaging the bacteria only when they come in contact with the surface ofthe material.

The inventors of the present invention have recently developed apolyelectrolyte coating using poly-L-arginine (PAR) as polycation andhyaluronic acid (HA) as polyanion. Said coating was used as a powerfulsurface coating with antimicrobial properties and with immunomodulatoryproperties (Özçelik, H. et al., 2015, Adv. Health. Mat, 4: 20126-2036).However, in this strategy, an antimicrobial peptide was further added toefficiently kill concomitantly bacteria, yeast and fungi. Moreover, thepoly-L-arginine used was not monodisperse, but the commercial batch ofpoly-L-arginine used was composed of polymeric chains with differentchain lengths and a molecular weight of more than 70 000 (whichcorrespond to polypeptide chains having more than 400 arginineresidues).

Contrary to this, in context of the present invention, the inventorsselected well-defined poly-L-arginine chains having a short chain lengthfrom 2 to 10 residues (PAR2 to PAR10) or 30 residues (PAR30), to builduplayer-by-layer coatings with HA as polyanion.

The inventors surprisingly demonstrated that the coatings comprisingpoly-L-arginine with a chain length of 10 and HA as polyanion showed astrong inhibition of bacterial growth of S. aureus for coatings havingmore than 25 bilayers, for example, 48 layers. These results areunexpected and surprising, in particular, because initial results withcoatings comprising PAR10, HA as polyanion and only 24 bilayers did notshow biocidal activity under the conditions tested.

The inventors further demonstrated, as a proof of principle, biocidalactivity against M. luteus, for coatings comprising PAR30, HA aspolyanion and 24 bilayers The biocidal activity against M. luteus (seeFIG. 11) is furthermore surprising and unexpected, because M. luteus ishyaluronidase deficient. The inventors thus demonstrated that thebiocidal activity of the polyelectrolyte coating of the invention isindependent of the degradation of the HA layer, which is contrary toprior art coatings that require the degradation of the HA layer.

Furthermore, contrary to other coatings, the mechanism responsible forthe biocidal activity of the coating of the present invention seems tobe based on the surface contact of the bacteria with the coating.

The inventors further demonstrated that the short length polycationmolecules, such as PAR10 and PAR30, diffuse within the coating and thebiocidal activity seems to depend on free diffusion of the polycationmolecules, because cross-linking of the coating reduces its biocidalfunction (see FIGS. 9 and 10).

SUMMARY OF THE INVENTION

The present invention therefore relates to a polyelectrolyte coating,comprising:

(a) at least one polycationic layer consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, and(b) at least one polyanionic layer consisting of hyaluronic acid.

The present invention also relates to a device comprising thepolyelectrolyte coating of the invention.

The present invention further relates to the use of the polyelectrolytecoating of the invention for producing a device of the invention, inparticular a medical device, more particularly an implantable device.

The invention further concerns a method for preparing a devicecomprising the polyelectrolyte coating of the invention, the methodcomprising:

(a) providing a device;(b1) depositing on the surface of said device(i) at least one polycationic layer consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, and thenii) at least one polyanionic layer consisting of hyaluronic acid, or(b2) depositing on the surface of said device ii) and then i) as definedabove, and optionally repeating step b1) and/or b2).

In a further aspect, the invention refers to a kit comprising

a) at least one polycationic material consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, andb) at least one polyanionic material consisting of hyaluronic acid.

In a further aspect, the invention refers to a method of preventing abacterial infection in an individual undergoing an implantation of animplantable device comprising the steps of providing an implantabledevice as defined herein below, and implanting said implantable devicein the individual wherein said implantable device prevents a bacterialinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Graph demonstrating the buildup of (PAR/HA) multilayer coatingon a SiO₂ coated crystal followed by QCM. Various molecular weight ofPAR (10, 30, 100 or 200 residues corresponding to notation PAR10 PAR30,PAR100 or PAR200 respectively) are used in association with HA.Evolution of the normalized frequency −Δfv/v (for v=3) as a function ofthe number of layers adsorbed. An exponential growth of the normalizedfrequency with the number of deposition step was observed for coatingsbuildup with PAR30, PAR100 or PAR200. The most important growth wasmonitored for larger PAR chains. In the case of PAR10 the increment inthe normalized frequency with the deposition number is the weaker,however an exponential growth was already observed.

FIG. 2: Image showing the confocal microscopy images of PAR/HA coatingsections (x,z). Observation by confocal microscopy of PAR/HA coatingsections (x,z) for PAR/HA coating buildups of (PAR/HA) multilayercoating on a SiO₂ coated crystal followed by QCM. The Buildup of(PAR/HA) multilayer coating with PAR of various molecular weight wascompared, i.e. PAR10 PAR30, PAR100 or PAR200. This image indicates thatthe obtained coatings were homogenously deposited on the surface in allconditions (for PAR with 10 to 200 residues).

FIG. 3: Graph demonstrating the normalized pathogen growth of S. aureusas a function of PAR concentration (mg*mL⁻¹) measured in solution. PARwith 10, 30, 100 or 200 arginine residues per chain were tested. EachPAR was incubated 24 h at 37° C. in 300 μL of MHB medium with S. aureus(A620=0.001). Pathogen growth of 0% corresponds to medium withantibiotics (and without PAR) and 100% to medium without PAR. Each valuecorresponds to the mean value of 3 individual experiments (3 samples perexperiment and condition) and error bars correspond to standarddeviations. For each concentration, the first column represents thenormalized pathogen growth of S. aureus for PAR10, the 2^(nd) column forPAR30, the third column for PAR100 and the fourth for PAR200.

FIG. 4: Graph demonstrating the buildup of (PAR10/HA)₁₀ multilayercoating on a SiO₂ coated crystal followed by QCM. PAR10 is used inassociation with HA. Evolution of the normalized frequency −Δfv/v (forv=3) as a function of the number of layers adsorbed. The increment inthe normalized frequency with the deposition number demonstrates alreadyan exponential growth.

FIG. 5: Graph demonstrating the buildup of (PAR/HA) multilayer coatingon a SiO₂ coated crystal followed by QCM. Various molecular weight ofPAR (10, 30, 100 or 200 residues corresponding to notation PAR10 PAR30,PAR100 or PAR200 respectively) are used in association with HA.Evolution of the estimated thickness as a function of the number ofadsorbed layers. An exponential growth of the estimated thickness withthe number of deposition step was observed for coatings buildup withPAR10, PAR30, PAR100 or PAR200 (the dark line on the top representPAR200, then light grey, PAR100, then the third line from the top PAR30,and the black line on the bottom represents PAR10). The most importantgrowth was monitored for larger PAR chains. In the case of PAR10 theincrement in the estimated thickness with the deposition number isweaker, however an exponential growth was already observed.

FIG. 6: Graph demonstrating the growth inhibition of S. aureus usingdifferent PAR coatings with 24 bi-layers. The normalized S. aureusgrowth (%) obtained in a supernatant after 24 h in contact with(PAR/HA)₂₄ multilayer coatings composed of poly-L-arginine with variousnumber of residues is shown. Each value corresponds to the mean value of3 experiments and error bars correspond to standard deviations. Thegrowth of S. aureus is about 80% for (PAR10/HA)₂₄, 75% for(PAR100/HA)₂₄, about 90% for (PAR200/HA)₂₄ and less than 5% for(PAR30/HA)₂₄ thus showing a strong growth inhibition of more than 95%for (PAR30/HA)₂₄.

FIG. 7: Graph demonstrating normalized fluorescence intensity of aphotobleached area according to [t(s)]_(1/2) for different PAR coatings.Different films (PAR10-FITC/HA)₂₄, (PAR30-FITC/HA)₂₄, (PAR100-FITC/HA)₂₄and (PAR20-FITC/HA)₂₄ are studied by using fluorescence recovery afterphotobleaching (FRAP) method. t=0 corresponds to the end of thephotobleaching step. Accordingly, the evolution of the normalizedfluorescence in the bleached area is demonstrated as a function of thesquare root of time. Recovery of fluorescence appears very fast forPAR10 (2^(nd) line from top of the graph) and PAR30 (1^(st) line fromtop of the graph) compare to PAR100 (3^(rd) line from top of the graph)or PAR200 (4^(th) line from top of the graph).

FIG. 8: Graph demonstrating the mobility of the different PAR chains.Proportion of mobile PAR (%) is estimated from data in FIG. 7. PAR10 andPAR30 chains are more mobile (between 85 to 90% of mobile fraction) thanthe PAR100 (only 63% of mobile fraction). The largest chains PAR200correspond to the slowest with about 12% of the population which ismobile.

FIG. 9: Graph demonstrating the mobility of PAR in (PAR30/HA)₂₄ orcrosslinked (PAR30/HA)₂₄. (PAR30/HA)₂₄ was cross-linked using EDC/NHSwith 0.5 M EDC and 0.1M NHS for 15 h at 4° C. Unreacted carboxyl groupswere neutralized using ethanolamine. The Proportion of mobile PAR (%) isestimated for (PAR30-FITC/HA)₂₄ compared to (PAR30-FITC/HA)₂₄ that hasbeen cross-linked with EDC-NHS. The proportion of mobile chains measuredby FRAP method decreases significantly from 88% for the non-cross-linkedfilm to 20% for the cross-linked one

FIG. 10: Graph demonstrating the growth inhibition of S. aureus using(PAR30/HA)₂₄ or crosslinked (PAR30/HA)₂₄. (PAR30/HA)₂₄ was cross-linkedusing EDC/NHS with 0.5 M EDC and 0.1M NHS for 15 h at 4° C. Unreactedcarboxyl groups were neutralized using ethanolamine. The normalized S.aureus growth (%) was measured in a supernatant after 24 h in contactwith (PAR30/HA)₂₄ or crosslinked (PAR30/HA)₂₄ multilayer coatings. Thegrowth of S. aureus is about 65% for crosslinked (PAR30/HA)₂₄ and lessthan 5% for (PLL30/HA)₂₄ thus showing that crosslinking reduces thebiocidal activity of the coating.

FIG. 11: Graph demonstrating the growth inhibition of different bacteriausing (PAR30/HA)₂₄ Normalized Growth of bacteria after 24 h in contactwith glass covered with the coating (PAR30/HA)₂₄. Bacteria growth isinhibited by more than 90% for S. aureus, methilicin resistant S.aureus, M. Luteus, E. Coli and P. aeruginosa.

FIG. 12: Graph demonstrating the growth inhibition of S. aureus overtime using (PAR30/HA)₂₄. The normalized S. aureus growth (%) obtained ina supernatant after 1 day, 2 days and three days in contact with(PAR30/HA)₂₄ multilayer coating is shown. The coating was put in contactwith a fresh suspension of S. aureus after each 24 h. Each valuecorresponds to the mean value of 3 experiments and error bars correspondto standard deviations. The growth of S. aureus is less than 5% for(PAR30/HA)₂₄ after 1 and 2 days, thus showing a strong growth inhibitionof more than 95% for (PAR30/HA)₂₄ in the first 48 hrs. After the 3 daythe inhibitory activity is reduced.

FIG. 13: Graph demonstrating the growth inhibition of S. aureus usingdifferent PAR coatings with 48 bi-layers. The normalized S. aureusgrowth (%) obtained in a supernatant after 24 h in contact with(PAR/HA)₄₈ multilayer coatings composed of poly-L-arginine with variousnumber of residues is shown. Each value corresponds to the mean value of3 experiments and error bars correspond to standard deviations. Thegrowth of S. aureus is less than 10% for (PAR10/HA)₄₈ thus showing astrong growth inhibition of more than 90% for (PAR10/HA)₄₈, the growthof S. aureus is less than 2% for (PAR30/HA)₄₈ thus showing a stronggrowth inhibition of more than 98% for (PAR30/HA)₂₄, and the growth ofS. aureus is about 95% for (PAR100/HA)₄₈ and about 75% for(PAR200/HA)₄₈.

DETAILED DESCRIPTION OF THE INVENTION Polyelectrolyte Coating

The inventors of the present inventions have demonstrated that apolyelectrolyte coating with polyarginine as polycation and hyaluronicacid (HA) as polyanion is a powerful surface coating with biocidalproperties. The inventors demonstrated that, a polyelectrolyte coatingwith poly-L-arginine (PAR, in particular, 10 or 30 arginine residues)and hyaluronic acid (HA) as polyanion has strong biocidal activities.

The inventors further demonstrated that, a polyelectrolyte coating withpoly-arginine with 10 arginine residues and hyaluronic acid (HA) aspolyanion has strong biocidal activity when the polyelectrolyte coatingsconsist of more than 24 layers, such as 48 layers.

The wording “polyelectrolyte”, as known by the skilled in the art,refers to polymers whose repeating units bear an electrolyte group.Polycations and polyanions are both polyelectrolytes. Accordingly, thepolyanionic and polycationic layers in context of the invention may bereferred to as polyelectrolyte layers.

The word “at least” in “at least one polyarginine” herein refers to atleast 1, 2, 3, 4, 5, 6, 7, 8 or 9 polyarginines consisting of nrepetitive arginine units, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9,polyarginines, more preferably one, two or three polyargininesconsisting of n repetitive arginine units, for example one polyarginineconsisting of n repetitive arginine units.

It will be therefore understood that in embodiments, wherein the “atleast one” in “at least one polyarginine” refers to more than onepolyarginine the “at least one polycationic layer” in context of theinvention consists of a mixture of specific polyarginines, wherein thespecific polyarginines differ from each other in their chain length. Itwill be understood by the skilled in the art, that, for example, the atleast one polyarginine may refer to, for example, two polyarginines,wherein one polyarginine has a chain length of, for example, 5 (n=5) andthe other polyarginine has a chain length of, for instance, 10 (n=10).

As mentioned herein above, polyarginine is a polycation, it will betherefore understood by the skilled in the art, that a “at least onepolyarginine consisting of n repetitive arginine units” may as well bereferred to as “at least one polycation consisting of n repetitivearginine units” and is a positively charged polymer and can also bereferred to as “polycationic material”.

Accordingly, in one embodiment, the polycationic material of nrepetitive arginine units constitutes the at least one polycationiclayer of the polyelectrolyte coating of the invention.

As defined in context of the invention, the “n” of the “n repetitivearginine units” is an integer comprised between 2 and 10.

In a further embodiment, n is an integer comprised between 3 and 10, forexample, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, such as 9 or10, preferably between 4 and 10, more preferably 5 and 10.

In a further embodiment, n is an integer comprised between 2 and 10, forexample, 2 and 9, 2 and 8, 3 and 9, 3 and 8, 3 and 7, 4 and 7.

In a further embodiment, n is an integer that is 10 or smaller than 10,is 9 or smaller than 9, is 8 or smaller than 8, is 7 or smaller than 7,is 6 or smaller than 6.

In one embodiment, n is an integer selected from the group consisting of2, 3, 4, 5, 6, 7, 8, 9, 10, preferably, n is 5 or 10.

The “repetitive arginine unit” can also be called “structural unit” andherein refers to the amino acid arginine, also referred to as arginineresidue.

In one embodiment, the n repetitive arginine units polymerize via theformation of a peptide bond. Accordingly, n repetitive arginine unitsmay be referred to as polymer or polypeptide. According to the number ofn, the polypeptide may be referred to as di-, tri, tetra-, penta-,hexa-, hepta-, octa, ennea- and deca-peptide for n=2, 3, 4, 5, 6, 7, 8,9, 10, respectively.

A “peptide bond”, also called amide bond, is a covalent chemical bondformed between the carboxyl group (COOH) of one amino acid and the aminogroup (NH₂) of another amino acid, wherein one molecule of water isproduced.

“Arginine” is an α-amino acid that is used in the biosynthesis ofproteins. Polyarginine refers to a polymer of the structural unitarginine. Polyarginine refers to poly-L-, poly-D- or poly-LD-arginine.In context of the present invention, polyarginine refers, in particular,to poly-L-arginine (PAR).

“Poly-L-arginine” is a positively charged synthetic polymer (also calledpolycation) and is produced in the form of a salt with a counterion. Thecounter ion may be selected from, but is not limited to, hydrochloride,hydrobromide or trifluoracetate.

In one example, polyarginine is poly-L-arginine hydrochloride withCAS#26982-20-7.

In one example, poly-L-arginine (PAR) such as PAR10 (10 arginine (R),Mw=2.1 kDa, PDI=1) and PAR5 were purchased from Alamanda Polymers, USA.

Methods to obtain polypeptides having n repetitive units such aspolyarginine with for example n=10 arginine units are known to theskilled in the art and include ring-opening polymerization ofalpha-amino acid N-carboxyanhydrides (NCAs) followed by purification.Typically, the polypeptides are purified after polymerization byprecipitation in water or, for example, in an organic nonsolvent and,after amino acid side chain deprotection, by dialysis. All water-solublepolymers are finally lyophilized.

Methods to obtain copolymers having n arginine units are also known tothe skilled in the art.

In one preferred embodiment, the at least one polyarginine refers to onepolyarginine consisting of n repetitive units and is monodisperse, i.e.the polycationic material of which the polycationic layer consists ismonodisperse. It will be understood by the skilled in the art that ifthe polycationic material of which the polycationic layer consists ismonodisperse, the polycationic layer is as well monodisperse.Accordingly, in one embodiment, the polycationic layer in context of thepresent invention is monodisperse.

“Monodisperse” herein refers to a polymer consisting of the samemolecules having the same mass. Synthetic monodisperse polymer chainscan be made, for example, by processes such as anionic polymerization, amethod using an anionic catalyst to produce chains that are similar inlength, for example, ring-opening polymerization of alpha-amino acidN-carboxyanhydrides (NCAs). It can be concluded on the basis of thepolydispersity index (PDI) if a sample of a polymer is monodisperse.Accordingly, in one embodiment, monodispersity is expressed using thepolydispersity index (PDI).

In some embodiments, when the at least one polyarginine is more than onepolyarginine, then the at least one polyarginine may be polydisperse,i.e. the polycationic material of which the polycationic layer consistsis a mixture of different polycations and may be polydisperse. It willbe understood by the skilled in the art that if the polycationicmaterial of which the polycationic layer consists is polydisperse, thepolycationic layer is as well polydisperse. Accordingly, in oneembodiment, the polycationic layer in context of the present inventionis polydisperse.

“Polydisperse” herein refers to a polymer consisting of differentmolecules having a different mass.

The “polydispersity index (PDI)” or “heterogeneity index”, or simply“dispersity”, is a measure of the distribution of molecular mass in agiven polymer sample. The polydispersity index (PDI) is calculated bydividing the weight average molecular weight (M_(w)) with the numberaverage molecular weight (M_(n)). The PDI has a value equal to orgreater than 1, but as the polymer chains approach uniform chain length,the PDI approaches 1.

Accordingly, in one embodiment, the polydispersity index (PDI) issmaller than 1.5, smaller than 1.4, smaller than 1.3, in particularbetween 1 and 1.2, preferably between 1 and 1.1, for example between 1and 1.05.

In one particular embodiment, the polydispersity index (PDI) of thepolyarginine, the polycationic material or the polycationic layer issmaller than 1.5, smaller than 1.4, smaller than 1.3, in particularbetween 1 and 1.2, preferably between 1 and 1.1, for example between 1and 1.05.

As known by the skilled in the art the PDI may be measured bysize-exclusion chromatography (SEC), light scattering measurements suchas dynamic light scattering measurements or mass spectrometry such asmatrix-assisted laser desorption/ionization (MALDI) or electrosprayionization mass spectrometry (ESI-MS).

In one example, the PDI is measured either on the protected polyaminoacids by, typically, gel permeation chromatography (GPC) in, forexample, DMF with 0.1M LiBr at typically 60° C. or on the deprotectedpolypeptides by, for example, GPC in typically aqueous buffer using, inboth cases, a calibration curve that was constructed from narrowpolydispersity PEG standards or universal calibration of TALLS. Theaverage molecular weight is provided by TALLS or by proton NMRspectroscopy using the amino acid repeating unit to incorporatedinitiator peaks integration ratio.

“Hyaluronic acid (HA)” also known as Hyaluronan is a linear (unbranched)polysaccharide or non-sulfated glycosaminoglycan, composed of repeatingdisaccharide units of N-acetyl glucosamine and glucuronate (linked by β1-3 and β 1-4 glycosidic bonds). Hyaluronic acid (HA) thus is anegatively charged polymer (also called polyanion) and is therefore, incontext of the present invention, also referred to as polyanionicmaterial. Said negatively charged polymer therefore exists together witha couter ion in form of a salt. For sodium hyaluronate the counterion issodium. It is distributed widely throughout connective, epithelial andneural tissues as part of the extra-cellular matrix. There are highconcentrations in the vitreous and aqueous humor of the eye, synovialfluid, skin, and the umbilical cord (Wharton jelly). The average 70-kgman has roughly 15 grams of hyaluronan in his body, one-third of whichis turned over (degraded and synthesized) every day. It is anevolutionarily conserved molecule being found in both the group A and CStreptococci and Pasteurella multocida as well as birds, mammals, andother orders of animals. In solutions of moderate to high molecularweight (500,000 to >3 million Da) at low concentrations it impartsconsiderable viscosity to aqueous solutions. Hyaluronic acid can bedegraded from Hyaluronidase. The molecular weight (Mw) of hyaluronanrepresents an average of all the molecules in the population and thusrepresents the molecular Mass Average (Molecular Weight Average). In oneexample, Hyaluronic acid has a molecular weight of 150 kDa and isbrought in form of Sodium Hyaluronate from Lifecore Biomed, USA.

“Hyaluronidase” is a family of enzymes that degrade hyaluronan. Theenzyme is found in most animal species and many micro-organisms. Thereare a number of different types of hyaluronidase with differentspecificity and kinetics.

In one embodiment, the polyelectrolyte coating of the invention mayfurther comprise a “pharmaceutical active drug”.

In the context of the present specification the term “pharmaceuticalactive drug” refers to compounds or entities which alter, inhibit,activate or otherwise affect biological events. For example, the drugincludes, but is not limited to, anti-cancer substances,anti-inflammatory agents, immunosuppressants, modulators ofcell-extracellular matrix interaction including cell growth inhibitors,anticoagulants, antrithrombotic agents, enzyme inhibitors, analgetic,antiproliferative agents, antimycotic substances, cytostatic substances,growth factors, hormones, steroids, non-steroidal substances, andanti-histamines. Examples of indication groups are, without beinglimited thereto analgetic, antiproliferativ, antithrombotic,anti-inflammatory, antimycotic, antibiotic, cytostatic,immunosuppressive substances as well as growth factors, hormones,glucocorticoids, steroids, non-steroidal substances, genetically ormetabolically active substances for silencing and transfection,antibodies, peptides, receptors, ligands, and any pharmaceuticalacceptable derivative thereof. Specific examples for above groups arepaclitaxel, estradiol, sirolimus, erythromycin, clarithromycin,doxorubicin, irinotecan, gentamycin, dicloxacillin, quinine, morphin,heparin, naproxen, prednisone, dexamethason.

In one embodiment, the “pharmaceutical active drug” is the polycationicmaterial as defined herein above.

In one embodiment, the polyelectrolyte coating of the invention isbiocompatible.

The term “biocompatible” as used in context of the invention, intends todescribe a coating that does not elicit a substantial detrimentalresponse in vivo.

As mentioned herein above, the inventors of the present inventiondemonstrated surprisingly that the polyelectrolyte coatings of theinvention have biocidal activity.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention has biocidal activity.

“Biocidal activity” herein refers to destroy, deter, render harmless, orexert a controlling effect on any harmful organism. A biocidal activityherein refers, for example, to an antimicrobial activity.

“Antimicrobial activity” herein refers to antiseptical, antibiotic,antibacterial, antivirals, antifungals, antiprotozoals and/orantiparasite activity, preferably antibacterial activity.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention has antibacterial activity and/or bacteriostatic activity.

In one embodiment the antibacterial activity and/or bacteriostaticactivity is directed against at least one bacterium.

“Bacteriostatic activity” herein refers to stopping bacteria fromreproducing, while not necessarily killing them, in other wordsbacteriostatic activity herein refers to inhibiting the growth ofbacteria. Accordingly, bacteriostatic activity may be expressed, forexample, in % of growth inhibition of at least one bacterium.

The “growth inhibition of at least one bacterium” in context of thepresent invention, may be more than 70%, for example, more than 75%,more than 80%, typically, more than 82, 84, 86, 88, 90, 91, 92, 93, 94,95, 96, 97, 98%. Accordingly, in one embodiment, the polyelectrolytecoating of the invention has more than 70% growth inhibition of at leastone bacterium, more particularly, more than 75%, more than 80%,typically, more than 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98%growth inhibition of at least one bacterium.

The “at least one bacterium” herein refers to bacteria of at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more species of bacteria.

In one particular example, the growth inhibition in context of thepresent invention, is measured by using S. aureus as at least onebacterium and the normalized S. aureus growth is measured after 24 h,after 18 h, or after 12 h, preferably 24 h contact with thepolyelectrolyte coating of the invention.

In one embodiment, the at least one bacterium is an ESKAPE pathogen.

The “ESKAPE pathogens” are the leading cause of nosocomial infectionsthroughout the world and are described in, for example, Biomed Res Int.2016; 2016: 2475067. In one embodiment, the term “ESKAPE pathogens”refers to a bacterium selected from the group constituted ofEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies.

In one embodiment, the at least one bacterium is a gram-positivebacterium or gram-negative bacterium, preferably gram-positivebacterium.

In one embodiment, the gram-negative bacterium is a Pseudomonasaeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia,Escherichia coli, Klebsiella pneumoniae, Enterobacter species orLegionella bacterium, preferably, Escherichia coli or Pseudomonasaeruginosa

In one embodiment, the gram positive bacterium is a Staphylococcus,Micrococcus or Enterococcus bacterium.

Bacteria of the “Staphylococcus” genus are stationary,non-spore-forming, catalase-positive, oxidase-negative, gram-positivecocci grouped together in grape-like clusters. Observed by Pasteur in1879 in furuncle pus, staphylococci owe their name to Ogsten (1881) whoisolated them in acute chronic abscesses. Bacteria of the“Staphylococcus” genus, such as, for example, S. aureus, S. epidermidis,S. capitis, S. caprae, S. haemolyticus, S. lugdunensis, S. schleiferi,S. simulans and S. warneri are the main agents of infections on foreignmaterials for example in prosthetic joint infections.

Accordingly, in one embodiment the Staphylococcus is selected from S.aureus, S. epidermidis, S. capitis, S. caprae, S. haemolyticus, S.lugdunensis, S. schleiferi, S. simulans and S. warneri, preferably S.aureus and S. epidermidis, more preferably S. aureus.

Bacteria of the “Micrococcus” genus are generally thought to be asaprotrophic or commensal organism, though it can be an opportunisticpathogen, particularly in hosts with compromised immune systems, such asHIV patients. Micrococci are normally present in skin microflora, andthe genus is seldom linked to disease. However, in rare cases, death ofimmunocompromised patients has occurred from pulmonary infections causedby Micrococcus. Micrococci may be involved in other infections,including recurrent bacteremia, septic shock, septic arthritis,endocarditis, meningitis, and cavitating pneumonia in particular inimmunosuppressed patients.

In one embodiment the Micrococcus is a M. luteus bacterium.

Bacteria of the “Enterococcus” genus are the cause of important clinicalinfections such as urinary tract infections, bacteremia, bacterialendocarditis, diverticulitis, and meningitis.

In one embodiment the Enterococcus is a vancomycin-resistantEnterococcus, such as E. faecalis or E. faecium.

The bacteriostatic activity or % of growth inhibition may bedemonstrated, for example, in an antibacterial assay as herein describedin the section “methods” herein below. Strains that may be used in suchan antibacterial assay may be, for example, M. luteus or S. aureus.

As mentioned in the introduction herein above, in one typicalapplication, the polyelectrolyte coating of the present inventionparticularly aims at preventing nosocomial infections related toimplants and medical devices. In said context, the risk of an infectionis especially high during the 6 hours post-implantation.

Accordingly, in one embodiment, the polyelectrolyte coating of thepresent invention has a biocidal activity, in particular anantibacterial activity and/or bacteriostatic activity, within the first24 hrs post implantation, for example within the first 12 hrs, first 9hrs, first 6 hrs post implantation.

As further mentioned in the introduction, antibacterial coatings have avast field of applications. Accordingly, in one embodiment thepolyelectrolyte coating of the invention has an anti-fouling activity.

“Fouling” or “Biofouling” or “biological fouling” herein refers to theaccumulation of microorganisms on a wetted surface.

“Anti-fouling” therefore herein refers to inhibiting the accumulation ofmicroorganisms on a wetted surface.

The polyelectrolyte coating of the present invention is typicallyconstructed using a layer-by-layer (LbL) deposition technique as furtherdescribed in the section “Method for preparing a device” herein below.Based on the layer-by-layer structure the polyelectrolyte coating mightalso be referred to as a “polyelectrolyte multilayer (PEMs)”,“polyelectrolyte film” or “polyelectrolyte matrix”.

Each of the polyelectrolyte layers has its given charge. Thepolycationic layer and the polyanionic layer, both form apolyelectrolyte network or a polyelectrolyte backbone. Thepolyelectrolyte layers attract each other by electrostatic interactions.Other attractive forces are based on hydrophobic, van der Waals, andhydrogen bonding interactions.

In general, during LbL deposition, a device, such as, for example, amedical device or an implantable device is dipped back and forth betweendilute baths of positively and negatively charged polyelectrolytesolutions. During each dip a small amount of polyelectrolyte is adsorbedand the surface charge is reversed, allowing the gradual and controlledbuild-up of electrostatically cross-linked polycation-polyanion layers.It is possible to control the thickness of such coatings down to thesingle-nanometer scale.

Accordingly, the polyelectrolyte coating of the invention typicallyincludes substantially ordered polyelectrolyte layers of alternatinglycharged polyelectrolyte layers.

In one embodiment, a single polyelectrolyte layer, such as onepolycationic or polyanionic layer, has a thickness of 1 nm to 10 nm. Thethickness of the polycationic and/or polyanionic layer depends on thecoating conditions and the polyelectrolyte material used.

In one embodiment, the polyelectrolyte coating has a thickness which istypically substantially thicker than the thickness of a singlepolyelectrolyte layer of the polyelectrolyte coating. In one embodiment,the polyelectrolyte coating may have a thickness of about 10 nm to about100 000 nm. The thickness of the polyelectrolyte coating depends on thecoating conditions, the number of, and the polyelectrolyte material usedfor, the polyelectrolyte layers.

The thicknesses of an obtained polyelectrolyte coating may be evaluated,for example, using confocal microscopy. Therefore, for example, 100 μLof PLL-FITC (poly-L-lysine labeled with fluorescein isothyocyanate, agreen fluorescent probe) (typically 0.5 mg·mL⁻¹ in Tris-NaCl buffer) aredeposited on top of a polyelectrolyte coating, for example a(PAR30/HA)₂₄ polyelectrolyte coating. After 5 minutes and diffusion ofPLL-FITC through the whole polyelectrolyte coating, a rinsing step istypically performed with Tris-NaCl buffer. Observations of the coatingsmay be carried out with a confocal microscope, such as Zeiss LSM 710microscope (Heidelberg, Germany) using a 20× objective (Zeiss, PlanApochromat).

As described above, the polyelectrolyte coating may be formed in aself-assembled manner to produce a Layer-by-Layer (LbL) structure.

The term “layer” in “polycationic layer” or “polyanionic layer” hereinrefers to an amount of polycations or polyanions as defined hereinabove, for example, deposited on, for example, the surface of a device,wherein said device is as defined herein below in the section “device”and may be, preferably, a medical device or an implantable device. As itwill be understood by the skilled in the art, in context of theinvention, one polycationic layer may consist of several layers of thesame polycationic material, and one polyanionic layer may consist ofseveral layers of the same polyanionic material. However, as it will befurther understood by the skilled in the art, even if one polycationicor polyanionic layer may consist of several layers, those layers will bereferred to as one layer in context of the present invention.Accordingly, the term polycationic layer as used in context of thepresent invention refers to the entire polycationic material that islocated between two polyanionic layers and the term polyanionic layer asused in context of the present invention refers to the entirepolyanionic material that is located between two polycationic layers.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” in context of the invention refers to at least25, at least 26, at least 28, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60, at least 65, at least70, at least 75, at least 80, at least 85, at least 90, at least 95, atleast 100, at least 200, at least 300, at least 400, at least 500polycationic layers and/or polyanionic layers.

In some embodiments “at least one polycationic layer” and/or “at leastone polyanionic layer” in context of the invention refers to at least25, at least 26, at least 28, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60 polycationic layersand/or polyanionic layers, preferably at least 30, at least 40, at least45 polycationic layers and/or polyanionic layers.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” refers to 30 to 500 polycationic and/orpolyanionic layers, for example 30 to 400, 30 to 300, 30 to 200, 30 to100, 30 to 90, 30 to 80, 35 to 80, 40 to 80, such as 40 to 75, 40 to 70,40 to 60 preferably 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60 layers.

In certain embodiments the number of the polycationic layers and thenumber of the polyanionic layers are the same.

In certain embodiments, the polycationic and polyanionic layers arealternating layers, in particular alternating charged polyelectrolytelayers.

Accordingly, in one embodiment, the polyelectrolyte coating comprises 30to 150 polycationic layers and/or 30 to 150 polyanionic layers,preferably the polyelectrolyte coating comprises 30 to 100 polycationiclayers and/or 30 to 100 polyanionic layers, more preferably, thepolyelectrolyte coating comprises 30 to 80 polycationic layers and/or 30to 80 polyanionic layers.

In one example, as further described herein below, it is possible tocover the surface of an object with one polycationic layer, to wash theobject and cover it again with one polycationic layer. These steps canbe repeated several times in order to obtain one polycationic layer of aspecific thickness which thus consists of several polycationic layers.As it will be understood by the skilled in the art this procedure can beused as well to obtain one polyanionic layer of a certain thicknesswhich is thus constituted of several polyanionic layers.

In one example, the polyelectrolyte coating consists of 30 layers ofpolyarginine having 10 arginine residues (PAR10) and 30 layers of HA,accordingly said coating will herein be called (PAR10/HA)₃₀. In the sameexample, the first layer is a polycation layer consisting of PAR10,followed by a first layer of the polyanion HA, followed by a secondpolycation layer consisting of PAR10 and followed by a second polyanionlayer consisting of HA. The layers are alternating until the 30^(th)polycation layer consisting of PAR10 and the 30^(th) polyanion layerconsisting of HA.

In another example, the polyelectrolyte coating consists of 48 layers ofpolyarginine having 10 arginine residues (PAR10) and 48 layers of HA,accordingly said coating will herein be called (PAR10/HA)₄₈. In the sameexample, the first layer is a polycation layer consisting of PAR10,followed by a first layer of the polyanion HA, followed by a secondpolycation layer consisting of PAR10 and followed by a second polyanionlayer consisting of HA. The layers are alternating until the 48^(th)polycation layer consisting of PAR10 and the 48^(th) polyanion layerconsisting of HA.

In a further aspect of the invention, the polyelectrolyte coating mayalso, for example, start with a polyanionic layer on the surface of anobject and finish with a polyanionic layer, in that case the number ofthe polycationic and polyanionic layers are different. Preferably, thepolyelectrolyte coating starts with a polyanionic layer, for instance,when the surface of the object is positively charged.

In another aspect of the invention, the polyelectrolyte coating mayalso, for example, start with a polycationic layer on the surface of anobject and finish with a polycationic layer; in that case the number ofthe polycationic and polyanionic layers is as well different.Accordingly, in certain embodiments the number of the polycationiclayers and the number of the polyanionic layers are different.Preferably, the polyelectrolyte coating starts with a polycationic layerwhen the surface of the object is negatively charged.

In one example, the polyelectrolyte coating consists of 49 layers ofpolyarginine having 10 arginine residues and 48 layers of HA,accordingly said coating will be called (PAR10/HA)₄₈/PAR10.

Accordingly, in certain embodiments the number of the polycationiclayers and the number of the polyanionic layers differ by one layer.

In certain embodiments, different polyelectrolyte layers consist of thesame polycationic material or of different polycationic materials asherein defined. For example, the polyelectrolyte coating may comprise 24layers consisting of polyarginine having 10 arginine residues and 24layers consisting of polyarginine having 5 arginine residues and 48layers consisting of HA.

The inventors of the present invention observed an exponential growth ofthe normalized frequency with the number of deposition steps for thebuild-up of the polyelectrolyte coatings with PAR10, PAR30, PAR100 orPAR200 and HA using quartz crystal microbalance (QCM), as furtherexplained in the examples and FIG. 1. The inventors further demonstratedthat the exponential increase of the thickness with the number ofdepositing step is related to the diffusion, in and out of the wholecoating, of at least one polyelectrolyte constituting the multilayer.The inventors further demonstrated using bleaching experiments that thepolycationic polymer contained in the coating is mobile and thusdiffuses inside the whole coating, as demonstrated in FIGS. 7 and 8.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention raises with an “exponential growth”, as called in theliterature, of the normalized frequency with the number of depositionsteps.

In a further embodiment, the “polyarginine consisting of n repetitivearginine units”, i.e. the individual peptide chains are mobile and/ordiffuse within the polyelectrolyte coating.

It has been shown by the inventors in context of PAR30, that thecovalent coupling of the at least two oppositely charged polyelectrolytelayers reduces the bacteriostatic activity of the coating. Since PAR30and PAR10 are similar in their mobility in the coating, it will beunderstood by the skilled in the art that this observation can betransferred to polyarginine having a shorter chain length. Accordingly,in one embodiment the at least one polycationic layer and the at leastone polyanionic layer are not covalently coupled.

Device

Staphylococcal infections on foreign material differ from conventionalinfections by the arrangement of bacteria in the form of biofilm.

“Biofilm” is a complex three-dimensional structure which is connected tothe foreign material and in which the bacteria cells are embedded in apolysaccharide extracellular matrix called slime or glycocalyx. Thisspecific structure may be formed by bacteria of the same species or ofdifferent species. In comparison with their living congeners in free (or‘planctonic’) form, these bacteria are in a state of quiescenceindicated by a low level of metabolic activity. Due to the reducedmetabolic activity bacteria in the form of a biofilm are, for example,more resistant to any antibacterial treatment.

The problematic of biofilms is not limited to the medical field.Biofilms are ubiquitous, occurring in aquatic and industrial watersystems as well as a large number of environments. Biofilms can avidlycolonize the surfaces of a wide variety of household items such astoilet bowls, sinks, toys, cutting boards, and countertops in kitchenand bathrooms. Biofilms may also occur in the food production industry,either in tins or on the machines that are used along the productionlines. At water and sewage treatment facilities, biofilms (biofouling)are also problematic: they cause metal corrosion, increased risk ofcontamination of products, decreased quality of water, and reducedefficacy of heat exchange for example for boards, and countertops inkitchen and bathroom.

Accordingly, an antibacterial coating is an advantage for devices inmany different applications.

As described above, the inventors of the present invention developed apolyelectrolyte coating having a biocidal activity. The inventorsdemonstrated for coatings comprising PAR10, in particular for(PAR10/HA)₄₈, that the polyelectrolyte coating has a biocidal activity,in particular within the first 24 hours when contacted with a solutioncontaining bacteria, more preferably the first 12, first 6 hours whencontacted with a solution containing bacteria, such as S. aureus.

The inventors further demonstrated for coatings comprising PAR30, inparticular for (PAR30/HA)₂₄, that the polyelectrolyte has a biocidalactivity, in particular within the first 72 hours when contacted with asolution containing bacteria, in particular within the first 48 hourswhen contacted with a solution containing bacteria, more preferably thefirst 24, first 12, first 6 hours when contacted with a solutioncontaining bacteria.

The polyelectrolyte coating of the present invention therefore inhibitsgrowth of a bacterium and thus prevents bacteria from the formation of abiofilm of the surface of a device comprising the polyelectrolytecoating of the invention.

The inventors demonstrated, for example, that (PAR/HA)₂₄ coatings builtwith PAR30 after 24/48 or 72 h of incubation show a total inhibition ofbacteria which demonstrate their efficiency over 3 days and threesuccessive contaminations.

The inventors further discovered that the time period of the biocidalactivity of the coatings of the invention increases with the number oflayers.

Accordingly, the time period of the biocidal activity of the coating ofthe present invention increases with the number of layers used.

The inventors further demonstrated that neither drying nor sterilizationof the coatings of the invention did modify the antimicrobial activityof the coating.

Accordingly no change in the total bactericide activity was measuredeven after sterilization.

It will be therefore be understood by the skilled in the art that due tothe bactericide activity of the polyelectrolyte coating of theinvention, said polyelectrolyte coating is particularly suitable forproducing a device comprising said polyelectrolyte coating.

Accordingly, the present invention further refers to a device comprisinga polyelectrolyte coating of the invention.

A “device” herein refers to an object comprising at least one surface.

In one embodiment, the polyelectrolyte coating covers at least a portionof the surface of said device.

In one embodiment, the surface of the device of the present inventioncomprises, consists of, or at least partly consists of metal such astitanium, plastic such as silicone, ceramic or other materials such aswood.

In a further embodiment, the surface of the device of the presentinvention exists in any kind of architecture depending on the materialused, accordingly, it will be understood by the skilled in the art, thatthe surface may be a flat surface, or uneven surface, such an unevensurface exists, for example, on surfaces of porous materials such astypically foams or fibers. In some examples, the surface may alsocomprise or consist of micro or nanoparticles.

Since the device comprises the polyelectrolyte coating of the invention,it will be understood by the skilled in the art, that, therefore, thefeatures associated with said polyelectrolyte coating that are furtherdefined above in the section “polyelectrolyte coating” also refer tosaid device.

Accordingly, in one embodiment, the device of the invention has biocidalactivity, wherein the biocidal activity is as defined in the section“polyelectrolyte coating” herein above.

In one aspect of the invention, the device of the invention has morethan 70% growth inhibition of at least one bacterium, more particularly,more than 75%, more than 80%, typically, more than 82, 84, 86, 88, 90,91, 92, 93, 94, 95, 96, 97, 98% growth inhibition of at least onebacterium. % growth inhibition of at least one bacterium is as definedherein above in the section “polyelectrolyte coating”.

In one embodiment, the growth inhibition is preferably measured with S.aureus wherein a solution inoculated with S. aureus is contacted withthe polyelectrolyte coating for 24 hours.

In a further embodiment, the device of the present invention has abiocidal activity or bacteriostatic activity within the first 72 hourspost implantation, for example within the first 48 hours, 24 hours, 12hours, first 9 hours, and first 6 hours post implantation.

In a preferred embodiment, the device of the present invention has abiocidal activity or bacteriostatic activity within the first 24 hourspost implantation, for example within the first 12 hours, first 9 hours,and first 6 hours post implantation.

Furthermore, in one embodiment the device of the invention hasanti-fouling activity. In a further embodiment, the device of theinvention has antibacterial activity and/or bacteriostatic activity.

Accordingly, in one embodiment, the device comprising a polyelectrolytecoating of the invention is a bacteriostatic device and/or a foulingresistant device. Bacteriostatic is as defined herein above in thesection “polyelectrolyte coating”.

The polyelectrolyte coating of the invention and the device of theinvention can be easily sterilized and stored without detrimentaleffects to the coating and its properties.

It will be further understood by the skilled in the art that due to thebiocidal and/or bacteriostatic activity of the polyelectrolyte coatingof the inventions, said polyelectrolyte coating is, in one embodimentparticularly suitable for producing a medical device, preferably animplantable device.

A “medical device” herein refers to an instrument, apparatus, implement,machine, contrivance, implant, in vitro reagent, or other similar orrelated article, including a component part, or accessory which is forexample intended for use in the diagnosis of disease or otherconditions, or in the cure, mitigation, treatment, or prevention ofdisease, in an individual as defined herein below, or intended to affectthe structure or any function of the body of an individual, and whichdoes not achieve any of its primary intended purposes through chemicalaction within or on the body of man or other animals and which is notdependent upon being metabolized for the achievement of any of itsprimary intended purposes. In one example, a medical device refers to adevice used for wound healing, such as bandages, for example adhesivebandages or dressings. In a further example, a medical device refers tosanitary articles.

A medical device herein may be destinated for use in an individual.

The term “individual”, “patient” or “subject” refers to a human ornon-human mammal, preferably a mouse, cat, dog, monkey, horse, cattle(i.e. cow, sheep, goat, buffalo), including male, female, adults andchildren.

Accordingly, in one embodiment, the medical device in context of theinvention may be a medical instrument.

In one embodiment, a medical instrument may be selected from the groupconsisting of a percussion hammer, pleximeter, thermometer, foreign bodydetector, stethoscope, specula, forceps, otoscope, or any accessoriesthereof, probes, retractors, scalpel, surgical scissors, boneinstruments, sharps spoons, suture needles and wounds clips.

In one embodiment, the medical device is an implantable device.

The “implantable device” of the present invention refers to a piece ofequipment or a mechanism that is placed inside of the body of anindividual to serve a special purpose or perform a special function.

An implantable device, in context of the present invention, may be, forexample, a prosthetic device or, for example, a device implanted todeliver medication, nutrition or oxygen, monitor body functions, orprovide support to organs and tissues. Implantable devices may be placedpermanently or they may be removed once they are no longer needed. Forexample, stents or hip implants are intended to be permanent. Butchemotherapy ports or screws to repair broken bones can be removed whenthey no longer needed.

In one embodiment, the implantable device is selected from the groupcomprising catheters, arteriovenous shunts, breast implants, cardiac andother monitors, cochlear implants, defibrillators, dental implants,maxillofacial implants, middle ear implants, neurostimulators,orthopedic devices, pacemaker and leads, penile implants, prostheticdevices, replacement joints, spinal implants, voice prosthesis,artificial hearts, contact lenses, fracture fixation device, infusionpumps, intracranial pressure device, intraocular lenses, intra-uterinedevices, joint prosthesis, prosthetic valves, orthopedic devices, suturematerials, urinary stents, vascular assist device, vascular grafts,vascular shunts and vascular stents, and artificial vessels of permanentor transient types.

In a preferred embodiment, the implantable device is selected from thegroup comprising catheters, defibrillators, prosthetic devices,prosthetic valves, replacement joints, orthopedic devices, pacemakers,vascular grafts, vascular shunts, vascular stents and intra-uterinedevices, preferably catheters, orthopedic devices, pacemakers andprosthetic devices.

A “catheter” herein refers to a tubular medical device for insertioninto canals, vessels, passageways, or body cavities for diagnostic ortherapeutic purposes, fluids and medication, but also in drainage ofbody fluids such as urine or abdominal fluids; angioplasty, angiography,and catheter ablation; administration of gases such as oxygen andvolatile anesthetic agents and hemodialysis.

A “shunt” typically refers to a narrow metal or plastic tube thatdiverts blood from one part to another.

A “stent” typically refers to a short narrow metal or plastic tube oftenin the form of a mesh that is inserted into the lumen of an anatomicalvessel as an artery or bile duct especially to keep a previously blockedpassageway open.

A “prosthetic valve” is for example a prosthetic heart valve.

Method for Preparing a Device of the Invention

The present invention further refers to a method for preparing a deviceof the invention herein referred to as the method of the invention.

The device of the invention may comprise different materials asspecified herein above, accordingly the at least one surface of thedevice to be covered with the polyelectrolyte coating may comprisedifferent materials as specified herein above. It will be understood bythe skilled in the art that said materials differ in their surfacecharge. Positively charged surfaces are, for example, the surfacesconsisting of amine group based polymers. Negatively charged surfacesare for example the surfaces consisting of carboxylic groups basedpolymers. It will be further understood by the skilled in the art, thata positively charged surface will be first covered with a polyanioniclayer and then with a polycationic layer, wherein a negatively chargedsurface will be first covered with a polycationic layer and then with apolyanionic layer. The two consecutive deposition steps may then berepeated as often as needed.

In one example, a device of the invention is prepared by depositing on,for example, its SiO₂ surface 48 bilayers of PAR/HA (PAR10/HA)₄₈ using,for example, an automated dipping robot, such as the an automateddipping robot of Riegler & Kirstein GmbH, Berlin, Germany. Therefore,the surface of the device is typically first washed with, for example,Hellmanex® II solution at 2%, H₂O, and ethanol and dried with air flow.Solutions of polyelectrolytes such as PAR and HA are prepared, forexample, by dissolving PAR and HA at typically 0.5 mg·mL⁻¹ in buffercontaining typically 150 mM NaCl and, for example, 10 mM oftris(hydroxymethyl)-aminomethane (TRIS, Merck, Germany) at, typically,pH 7.4. The surface of the device is dipped alternatively in polycationand polyanion solutions and extensively rinsed in NaCl-Tris bufferbetween each step. After preparation, the coating is, typically, driedwith air flow and then immerged in NaCl-Tris buffer and stored at 4° C.before use.

The wording “depositing on the surface of said device at least onepolycationic layer” and “depositing on the surface of said device atleast one polyanionic layer” of step b1) i) and ii) or b2) ii) and i)herein refers to contacting the surface of said device with apolycationic solution in case of step b1) i) or b2 i) or to contactingthe surface of said device with a polyanionic solution in case of stepb1) ii) or b2 ii).

The “surface of said device” herein refers to at least one surface, saidat least one surface may be partially covered by the polyelectrolytecoating of the invention. The at least one surface is preferably onesurface.

A “polycationic solution” or “polyanionic solution” herein refers to asolution comprising polycationic material or polyanionic material asdefined herein above in the section “polyeletrolyte coating”. In contextof the present invention, typically a “polycationic solution” and a“polyanionic solution” might be referred to as polyelectrolyte solution.

In one example, the polycationic solution herein refers to a solutioncomprising at least one polyarginine consisting of n repetitive arginineunits, wherein n is an integer between 2 and 10.

In one example, the polyanionic solution herein refers to a solutioncomprising hyaluronic acid.

“Contacting” as herein used refers typically to spraying, immersing,dipping or pouring.

Accordingly, in one embodiment, for “depositing the polyelectrolytelayers according to step (b1) and/or (b2)”, a polyelectrolyte solutionmay, for instance, be sprayed onto the surface of a device on which thepolyelectrolyte coating is to be formed.

Alternatively, or in combination, the surface of said device can beimmersed or dipped into a polyelectrolyte solution or a polyelectrolytesolution can be poured onto the surface of the substrate.

Accordingly, in one embodiment, the at least one polycationic layer isdeposited on the surface of the device in step b1) i) by contacting thesurface of said device with a polycationic solution comprising at leastone polyarginine consisting of n repetitive arginine units as definedherein above.

Accordingly, in one embodiment, the at least one polyanionic layer isdeposited on the surface of the device in step b1) ii) by contacting thesurface of said device with a polyanionic solution comprising HA.

It will be understood that the steps b1) and/or b2) may be repeated asoften as necessary by the skilled in the art in order to obtain a devicecomprising a polyelectrolyte coating with the number of polyelectrolytelayers, in particular alternating polycationic and polyanionic layers,as defined herein above in the section “polyelectrolyte coating”. Itwill be clear to the skilled in the art, that the deposition of thepolyelectrolytes may be influenced by the pH.

Accordingly, in one embodiment, said “polycationic solution” and/or“polyanionic solution” in context of the invention may further comprisea buffer.

A “buffer” is an aqueous solution consisting of a mixture of a weak acidand its conjugate base or a weak base and its conjugate acid. Its pHchanges very little when a small amount of strong acid or base is addedto it and thus it is used to prevent changes in the pH of a solution.Buffer solutions are used as a means of keeping pH at a nearly constantvalue in a wide variety of chemical applications. Buffers used in thecontext of the invention might be, for example, PBS (Phosphate bufferedsaline) or TRIS (tris(hydroxymethyl)aminomethane).

In one embodiment, said “polycationic solution” and/or “polyanionicsolution” in context of the invention has pH ranging from 4 to 9,preferably 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 and typically, a pHranging from 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, preferably 7 to8, in particular 7, 7.2, 7.4, 7.6, 7.8, 8, for example 7.4.

It will be further clear to the skilled in the art, that the depositionof the polyelectrolytes may be influenced by the interaction between thecharged polyelectrolytes and the charged surface onto which thepolyelectrolytes are to be deposited, and the interaction between thecharged polyelectrolytes among each other. These interactions can be atleast partially controlled by the ion strength of the solution.

Therefore, in some embodiments the ion strength of the polyelectrolytesolution, used in step (b1) or (b2) of the method of the invention isadjusted in certain embodiments to increase the amount of the depositedpolyelectrolytes.

Accordingly, in one embodiment, said “polycationic solution” and/or“polyanionic solution” in context of the invention may further compriseions.

An “ion” is an atom or molecule in which the total number of electronsis not equal to the total number of protons, giving the atom or moleculea net positive (cation) or negative (anion) electrical charge. An ionconsisting of a single atom is an atomic or monatomic ion; if itconsists of two or more atoms, it is a molecular or polyatomic ion. Ionsare further distinct according to the charge they carry. Therefore anion can be a monovalent ion or bivalent (sometimes called divalent ion)or polyvalent ions. Ions may be without limitation F⁻, Cl⁻, I⁻, NH₄ ⁺,SO₄ ²⁺, Ca²⁺, Mg²⁺, Na⁺.

Typically, ions may be added in the form of a buffer, as defined hereinabove, or in the form of a salt, such as for example NaCl.

For example, solutions of polyelectrolytes such as PAR and HA weretypically prepared, for example, by dissolving PAR and HA at typically0.5 mg·mL-1 in sterilized buffer containing typically 150 mM NaCl and,for example, 10 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck,Germany) at, typically, pH 7.4.

Between the depositions of the polyelectrolyte layers at least onewashing or also called rinsing step can be performed in a solutionwithout polyelectrolytes to remove not-assembled polyelectrolytematerial.

Accordingly, in one embodiment, the method of the invention furthercomprises at least one washing step after step i) and/or ii) of b1) orb2).

In one embodiment, for washing, a solution without polyelectrolytes maybe used. Said solution may be water or any other solution that seemssuitable to the skilled in the art. In one embodiment, washing isperformed using buffer, for example NaCl-Tris buffer. In one example,said NaCl-Tris buffer comprises 150 mM NaCl and 10 mM Tris at a pH 7.4.

Alternatively, or in addition to that, in one embodiment, a drying stepor steps can be performed.

Accordingly, in one embodiment, the method of the invention furthercomprises at least one drying step after step i) and/or ii) of b1) orb2) and/or the washing step of claim.

“Drying” may be performed by any method known to the skilled in the art,such as air heating, natural air drying, dielectric drying, air flow,preferably air flow. In one example air flow refers to purging withnitrogen gas.

In one embodiment, after a drying step, the same polyelectrolytesolution as used immediately before the drying step can be depositedagain on the surface since the drying can lead to a partial exposure ofbinding sides or charges of the underlying polyelectrolyte layer, saiddrying step may therefore also be called an intermediate drying step. Byapplying such a sequence, the load of a particular polyelectrolyte, canbe further increased.

Accordingly, in one embodiment when the method of the invention furthercomprises at least one drying step the previous step i) or ii) of b1) orb2) and/or the washing step may be repeated.

Typically, a polyelectrolyte solution is brought into contact with thesurface and the surface is allowed to dry for a given time, for examples10 to 60 sec by purging, for example, with nitrogen gas. Subsequently,the same polyelectrolyte solution is again brought into contact with thesurface.

In certain embodiments, at least one washing and/or drying step iscarried out between two consecutive deposition steps.

It has been shown by the inventors, that the covalent coupling of the atleast two oppositely charged polyelectrolyte layers reduces thebacteriostatic activity of the coating.

Accordingly, in one embodiment the method of the invention does notcontain a step to covalently couple the at least one polycationic layerwith the at least one polyanionic layer.

In one embodiment, the method of the invention further comprises a step(c) wherein the polyelectrolyte coating obtained in steps (a) and (b1)or (b2) of the method as defined above may be further soaked with apharmaceutical active drug. The pharmaceutical drug is as defined in thesection “Polyelectrolyte coating” herein above.

In one particular embodiment, the method for preparing a device is amethod for preparing an implantable device.

Accordingly, in one embodiment, the invention refers to a method forpreparing an implantable device comprising a polyelectrolyte coating,the method comprising:

(a) providing an implantable device;(b1) depositing on the surface of said implantable device(i) at least one polycationic layer consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, and thenii) at least one polyanionic layer consisting of hyaluronic acid, or(b2) depositing on the surface of said implantable device ii) and theni) as defined above, and optionally repeating step b1) and/or b2).

As described herein above under the section “device”, the surface of thedevice of the present invention comprises, consists of, or at leastpartly consists of metal such as titanium, plastic such as silicone,ceramic or other materials such as wood. These surfaces may be charged,such as positively or negatively charged. As a result, the skilled inthe art will therefore understand that a device comprising a surfacethat is, for example, negatively charged might be covered first with acationic layer.

In some embodiments, the device might undergo a surface treatment priorto the method for preparing an implantable device in order to have acharged surface.

Accordingly, the surface of the device provided in step a) might becharged, such as positively charged or negatively charged, wherein thesurface is as defined herein above in the section “device”.

Alternatively, in some embodiments the method for preparing animplantable device might comprise a step (a2) of charging the surface ofsaid implantable device.

The skilled in the art knows methods to charge the surfaces of a device,such methods include the adsorption of ions, protonation/deprotonation,UV radiations and the application of an external electric field. Theskilled in the art further knows, for example, that surface chargepractically always appears on a device surface when it is placed into afluid. Most fluids contain ions, positive (cations) and negative(anions). These ions interact with the device surface. This interactionmight lead to the adsorption of some of them onto the surface. If thenumber of adsorbed cations exceeds the number of adsorbed anions, thesurface would have a net positive electric charge, and if the number ofadsorbed anions exceeds the number of adsorbed cations, the surfacewould have a net negative electric charge.

Use

In one embodiment the invention refers to the use of the polyelectrolytecoating of the invention for producing a device of the invention, inparticular a fouling resistant device and/or bacteriostatic device.

In a further embodiment the invention refers to the use of thepolyelectrolyte coating of the invention for producing a medical deviceor an implantable device.

Kit

The invention further provides a kit comprising

a) at least one polycationic material consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, andb) at least one polyanionic material consisting of hyaluronic acid.

In particular, the invention further provides a kit comprising

a) at least one polycationic material consisting of one polyarginineconsisting of n repetitive arginine units, wherein n is an integercomprised between 2 and 10, andb) at least one polyanionic material consisting of hyaluronic acid.In one embodiment, the kit further comprises instructions regarding theuse of the polycationic and polyanionic material. These instructions maye.g. describe a method for preparing an implantable device as definedherein above.

The wording “at least” in “at least one polycationic material” or in “atleast one polyanionic material” herein refers to at least 1, 2, 3, 4, 5,6, 7, 8 polycationic materials.

The word “at least” in “at least one polyarginine” herein refers to atleast 1, 2, 3, 4, 5, 6, 7, 8 or 9 polyarginines consisting of nrepetitive arginine units, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9,polyarginines, more preferably one polyarginine consisting of nrepetitive arginine units.

Therapeutic Methods and Uses

Infections, such as Staphylococcal infections, on foreign materialassociated with biofilm formation have a number of features whichdistinguish them completely from conventional tissue infections. Theseinfections are most often paucibacillary (having few bacteria) andreadily polymicrobial. The bacteria have a very slow metabolism whichkeeps them in a state close to dormancy and the genes which they expressare different to those activated in planctonic forms. The state ofdormancy of the bacteria and the presence of the biofilm significantlyreduce the inflammatory reaction and the attraction of immune cells atthe infection site. Lastly, for the same reasons, the bacteria arelargely protected from the action of antibiotics. It is thereforeimportant to prevent said biofilm formation, in particular, in the bodyof an individual, for example after transplantation.

The polyelectrolyte coating or implantable device in context of thepresent invention have a biocidal activity, in particular within thefirst 72 hours post-implantation, in particular within the first 48hours post-implantation, more preferably the first 24, first 12, first 6hours post implantation. The polyelectrolyte coating or implantabledevice in context of the present invention therefore inhibit growth of abacterium and thus prevents them from the formation of a biofilm of thesurface of said coating or implantable device and thus prevents abacterial infection.

Accordingly, in one embodiment, the invention refers to a method ofpreventing a bacterial infection in an individual undergoing animplantation of an implantable device comprising the steps of providingan implantable device as defined herein above, and implanting saidimplantable device in the individual, wherein said implantable deviceprevents a bacterial infection.

In one embodiment, providing an implantable device refers to preparingan implantable device according to the method of the invention.

The individual is as defined herein above in the section “device”.

In one embodiment, the bacterial infection is a nosocomial infection.

A “nosocomial infection” also called Hospital-acquired infection (HAI)herein refers an infection that is contracted from the environment orstaff of a healthcare facility. The infection is spread to thesusceptible patient in the clinical setting by a number of means, inparticular by implantable devices. Gram positive bacteria involved in“nosocomial infection” are for example Staphylococcus aureus.Gram-negative bacteria involved in “nosocomial infection” are forexample Pseudomonas aeruginosa, Acinetobacter baumannii,Stenotrophomonas maltophilia, Escherichia coli, Legionella.

Accordingly, in one embodiment the nosocomical infection is an infectioncaused from at least one bacterium as defined above in the section“Polyelectrolyte coating” herein above.

As further specified in the section “device” bacterial infection in formof a biofilm differ from conventional infections.

Accordingly, in one embodiment, the bacterial infection is a biofilminfection.

A “biofilm infection” herein refers to a bacterial infection withbiofilm forming bacteria. Biofilm forming bacteria are, for example, butis not limited to, gram-positive bacteria such as Staphylococcus, andgram-negative species such as Escherichia coli, or Pseudomonasaeruginosa.

Any combination of the above embodiments makes part of the invention.

Throughout the instant application, the term “comprising” is to beinterpreted as encompassing all specifically mentioned features as welloptional, additional, unspecified ones. As used herein, the use of theterm “comprising” also discloses the embodiment wherein no featuresother than the specifically mentioned features are present (i.e.“consisting of”). Furthermore the indefinite article “a” or “an” doesnot exclude a plurality. The mere fact that certain measures are recitedin mutually different dependent claims does not indicate that acombination of these measures cannot be used to advantage.

The invention will now be described in more detail with reference to thefollowing examples. All literature and patent documents cited herein arehereby incorporated by reference. While the invention has beenillustrated and described in detail in the foregoing description, theexamples are to be considered illustrative or exemplary and notrestrictive.

EXAMPLES 1. Example 1

1.1 Material

Polyelectrolyte multilayer coatings have been built up with thefollowing polyelectrolytes. Polycations of poly(I-argininehydrochloride) (PAR) were purchased from Alamanda Polymers, USA.Different PAR polymers used differ from the numbers of arginine perchain: PAR10 10 arginine (R), Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200R, Mw=40.8 kDa, PDI=1.06). Hyaluronic acid (HA, Mw=150 kDa) used as thepolyanion was from Lifecore Biomed, USA.

1.2 Methods

Monitoring Build-Up of Multilayer Coatings:

Coating or film build-up was followed using an in situ quartz crystalmicrobalance (QCM-D, E1, Q-Sense, Sweden). The quartz crystal is excitedat its fundamental frequency (about 5 MHz), as well as at the third,fifth, and seventh overtones (denoted by v=3, v=5, v=7 correspondingrespectively to 15, 25, and 35 MHz). Changes in the resonancefrequencies (Δf) are measured at these four frequencies. An increase ofΔf/v is usually associated to an increase of the mass coupled with thequartz. PAR (i.e. PAR10, PAR30, PAR100 or PAR200) and HA were dissolvedat 0.5 mg·mL⁻¹ in sterilized buffer containing 150 mM NaCl and 10 mM oftris(hydroxymethyl)-aminomethan (TRIS, Merck, Germany) at pH 7.4. Thepolyelectrolyte solutions were successively injected into the QCM cellcontaining the quartz crystal and PAR was the first depositedpolyelectrolyte. They were adsorbed for 8 min and then, a rinsing stepof 5 min with NaCl-Tris buffer was performed.

Buildup of (PAR/HA)₂₄ with Dipping Robot:

For the construction of 24 bilayers of PAR/HA ((PAR30/HA)₂₄) anautomated dipping robot was used (Riegler & Kirstein GmbH, Berlin,Germany). Glass slides (12 mm in diameter) were first washed withHellmanex® II solution at 2%, H₂O, and ethanol and dried with air flow.The solutions of polyelectrolytes were prepared as described above forQCM experiments. Glass slides were dipped alternatively in polycationand polyanion solutions and extensively rinsed in NaCl-Tris bufferbetween each step. After construction, the coatings were dried with airflow and then immerged in NaCl-Tris buffer and stored at 4° C. beforeuse. Thicknesses of obtained coatings were evaluated by deposition of100 μL of PLL-FITC (poly-L-lysine labelled with fluoresceinisothyocyanate, a green fluorescent probe) (0.5 mg·mL⁻¹ in Tris-NaClbuffer) on top of the PAR/HA multilayer coatings. After 5 minutes anddiffusion of PLL-FITC through the whole coating, a rinsing step wasperformed with Tris-NaCl buffer. Observations of the coatings werecarried out with a confocal microscope Zeiss LSM 710 microscope(Heidelberg, Germany) using a 20× objective (Zeiss, Plan Apochromat).

24 bilayers of PLL/HA (PLL30/HA)₂₄ and 24 bilayers of PLO/HA (i.e.(PLO30/HA)₂₄, (PLO100/HA)₂₄ and (PLO250/HA)₂₄) were prepared in analogy,wherein PLO or PLL was dissolved at 0.5 mg·mL⁻¹ in sterilized buffercontaining 150 mM NaCl and 10 mM of tris(hydroxymethyl)-aminomethan(TRIS, Merck, Germany) at pH 7.4.

Other polyelectrolyte coatings, for example polyelectrolyte coatingscomprising 48 bilayers of PAR/HA such as (PAR30/HA)₄₈ and (PAR10/HA)₄₈were prepared in analogy.

Antibacterial Assays:

Staphylococcus aureus (S. aureus, ATCC 25923) strains were used toassess the antibacterial properties of the test samples. Bacterialstrain was cultured aerobically at 37° C. in a Mueller Hinton Broth(MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10mL of MHB medium and incubated at 37° C. for 20 h, to provide a finaldensity of 10⁶ CFU·mL⁻¹. To obtain bacteria in the mid logarithmic phaseof growth, the absorbance at 620 nm of overnight culture was adjusted of0.001.

Glass slides coated with (PAR/HA)₂₄ with PAR10, PAR30, PAR100, aresterilized by using UV-light during 15 minutes, then washed withNaCl-Tris buffer. After washing, each glass slides were deposited in24-well plates with 300 μl of S. aureus, A₆₂₀=0.001, and incubatedduring 24 hours at 37° C.

For negative control, uncoated glass slides were directly incubated withS. aureus using a similar method.

For positive control, Tetracycline (10 μg·mL⁻¹) and Cefotaxime (0.1μg·mL⁻¹) were added in S. aureus solution in contact with uncoated glassslides.

To quantify bacteria growth or inhibition after 24 h, the absorbance ofthe supernatant at 620 nm was measured.

The assay was performed similarly for Glass slides coated with(PLL30/HA)₂₄, (PLO30/HA)₂₄, (PLO100/HA)₂₄ and (PLO250/HA)₂₄.

The antibacterial assay for M. Luteus, E. Coli and P. aeruginosa wereperformed in analogy to the bacterial assay described for Staphylococcusaureus described above.

Bacteria Live Dead Assay:

To evaluate the health of bacteria which are on the surface, theBacLight™ RedoxSensor™ CTC Vitality Kit (ThermoFischer Scientific Inc.,France) was used. This kit provides effective reagents for evaluatingbacterial health and vitality. The kit contains 5-cyano-2,3-ditolyltetrazolium chloride (CTC), which has been used to evaluate therespiratory activity of S. aureus. Indeed, healthy bacteria will absorband reduce CTC into an insoluble, red fluorescent formazan product.Bacteria which are dead or with a slow respiratory activity will notreduce CTC and consequently will not produce red fluorescent product.Finally this kit gives a semi-quantitative estimate of healthy vsunhealthy bacteria. SYTO® 24 green-fluorescent nucleic acid stain(ThermoFischer Scientific Inc., France) is used for counting allbacteria. A solution of 50 mM CTC and 0.001 mM Syto 24 in pure water isprepared. Each glass slides were washed with phosphate-buffered salinebuffer, pH=7.4 (PBS) then 270 μl of PBS and 30 μL of CTC/Syto 24solution were added. The plate were incubated 30 minutes at 37° C., awayfrom light. Each surfaces were observed with confocal microscopy (ZeissLSM 710 microscope, Heidelberg, Germany), using a 63× objective,immerged in oil. Excitation/Emission wavelength of stains was 450/630 nmfor CTC and 490/515 nm for Syto 24.

Biocompatibility Test:

Human fibroblast (CRL-2522 from ATCC/LGC Standards, France) was culturedat 37° C. in Eagle's Minimum Essential Medium (EMEM, ACC/LGC) with 10%of Fetal Bovin Serum (FBS, Gibco/ThermoFicher Scientific Inc., France)and 1% of penicillin streptomycin (Pen Strep, LifeTechnologies/ThermoFicher Scientific Inc., France). 50 000 cells wereincubated in each well of a 24 well-plates during 24 h. Glass slidescoated with (PAR/HA)₂₄ were incubated simultaneously in a 6 well-plateswith 1 mL of medium. After 24 h, the medium of the wells containingcells was removed and replaced by the supernatant that was in contactwith the multilayers for 24 h. Human fibroblasts were incubated during24 h at 37° C. Then, the supernatant was removed and incubated with 10%of AlamarBlue (ThermoFischer Scientific Inc., France) during 2 h. Thecell viability was determined by measuring the fluorescence of producedresofurin (Excitation/Emission wavelength=560/590 nm). Cells were washedtwice with PBS and fixed with PFA 4% solution during 10 minutes, andthen again washed twice with PBS. A solution of Phalloidin was preparedin PBS buffer with 1% of bovin serum albumin (BSA). The stainingsolution were placed on the fixed cells for 30 minutes at roomtemperature and washed two times with PBS buffer. A solution of DAPI wasprepared and placed on the cells at the same conditions as previously.Fluorescence images were captured using Nikon Elipse Ti-S with 63×PL APO(1.4 NA) objective equipped with Nikon Digital Camera (DS-Q11MC withNIS-Elements software, Nikon, France), and processed with ImageJ(http://rsb.info.nih.gov/ij/). Excitation/Emission wavelength forRhodamine Phalloidin was 540/565 nm and for DAPI 350/470 nm.

Time-Lapse Microscopy:

Glass slides coated with (PAR/HA)₂₄ were sterilized by using UV-lightduring 15 minutes, then washed with NaCl-Tris buffer. After washing,each glass slides were mounted in a Ludin Chamber (Life ImagingServices, Switzerland) at 37° C., 5% CO₂, with 1 mL μl of S. aureus(used as described previously, with A₆₂₀=0.001), stained with Syto 24during the culture. The time-lapse sequence was performed during 24 hwith a Nikon TIE microscope equipped with a 60×PL Apo oil (1.4 NA)objective and an Andor Zyla sCMOS camera (Andor Technology LtD. UnitedKingdom), was used with Nikon NIS-Elements Ar software (Nikon, France).Phase contrast and fluorescence images were acquired every 5 min for 24h. Images were processed with ImageJ.

Circular Dichroism:

Circular dichroism (CD) spectra were recorded using a Jasco J-810spectropolarimeter (Jasco Corporation, UK) as an average of 3 scansobtained using a 0.1 mm path length quartz cuvette at 22° C. from 180 to300 nm with data pitch of 0.1 nm and a scan speed of 20 nm/min. Allspectra were corrected by subtraction of the buffer spectra. Spectra foreach PAR were obtained at a concentration of 2 mg·mL⁻¹ in NaCl-TrisBuffer. All CD data were expressed as mean residue ellipticity.

Fluorescent Labelling of PAR:

For labeling PAR chains, PAR (15 mg·mL⁻¹ in 100 mM NaHCO₃ pH 8.3 buffer)was incubated with fluorescein isothiocyanate (FITC, Sigma Aldrich,France) at 1:2 molar ratio of PAR/FITC at room temperature for 3 h. Thissolution was dialyzed against 1 L of water at 4° C. with a Slide-A-LyserDialysis Cassette (Thermo Fischer Scientific Inc, USA), cut-off=3500MWCO. PAR-FITC was then produced and stored in aliquots of 2 mL (0.5mg·mL⁻¹ in NaCl-Tris buffer).

Release Experiments:

For the first experiment, a multilayer coating (PAR30/HA)₂₄ was built byusing PAR-FITC. Release experiments were performed at 37° C. during 24 hin presence of MHB medium or a S. aureus/MHB solution (A₆₂₀=0.001). 300μL of mineral oil were added on the top of the supernatant to preventany evaporation during the monitoring. The release of PAR-FITC insolution was performed by measuring the fluorescence of the supernatantover time with a spectrofluorimeter (SAFAS Genius XC spectrofluorimeter,Monaco) with excitation/emission wavelength of 488/517 nm. Three sampleswere studied for each conditions.

For the second experiment, a multilayer film (PAR30/HA)24 was incubatedat 37° C. with two conditions: A) with 300 μl of S. aureus solutionA620=0.001 and B) 300 μl of MHB only. After 24 h, the supernatant incontact with the LbL was taken and incubated with a new S. aureussolution to have a final A620=0.001. After 24 h at 37° C., theabsorbance at 620 nm was measured. Three samples were studied for eachcondition.

Fluorescence Recovery after Photobleaching (FRAP) Experiments:

The diffusion coefficient, D, and the proportion of mobile molecules, p,was measured for (PAR/HA)₂₄ multilayers containing PAR-FITC byperforming photobleaching experiments (FRAP, Fluorescence Recovery AfterPhotobleaching).

A glass slide coated with the multilayer was introduced in a home-madesample holder and immerged in 200 μl of Tris-NaCl buffer. One circularregions (4.4 μm in radius and refered as “R4” in an image of 35 μm×35 μmor 10.6 μm in radius and refered as “R10” in an image of 85 μm×85 μm)were exposed for 700 msec to the laser light set at its maximum power(λ=488 nm). Then, the recovery of the fluorescence in the bleached areawas observed over time. Observations were carried out with a Zeiss LSM710 microscope (Heidelberg, Germany) using a 20× objective (Zeiss, PlanApochromat).

Cross-Linking of (PAR30/HA)₂₄:

Crosslinking was performed by immersing the (PAR30/HA)24 films in asolution containing EDC (100 mM) and N-hydroxysuccinimide (10 mM) inNaCl (0.15 M) during 15 h at 4° C. Films were rinsed 2 times with a NaCl(0.15 M) solution. The films were immerged in a solution of ethanolamine(1M) during 40 minutes at 4° C. to neutralize all carboxylates functionsthat have not react. The films were rinsed with NaCl solution and theNaCl-Tris buffer solution was used for the last rinsing step.

1.3 Results

In order to test the buildup of the PAR/HA coatings, quartz crystalmicrobalance (QCM) was used. FIG. 1 corresponds to the layer-by-layerdeposition monitored with QCM for various molecular weight of PAR (10,30, 100 or 200 residues corresponding to notation PAR10 PAR30, PAR100 orPAR200 respectively). In a first approximation, it is known that theincrease in the normalized frequency could be related to an increase inthe deposited mass or thickness (REF). An exponential growth of thenormalized frequency with the number of deposition step was observed forcoatings buildup with PAR30, PAR100 or PAR200. The most important growthwas monitored for larger PAR chains (FIG. 1). In the case of PAR₁₀ theincrement in the normalized frequency with the deposition number is theweaker, however an exponential growth was already observed (FIGS. 1 and4). Finally, despite the short length of this polypeptide, thelayer-by-layer growth was effective.

We also estimated the thicknesses of the films by using the model ofVoinova et al. (Phys. Scripta 1999, 59, 391-396). After 8 deposited pairof layers (or “bi-layers”) the thicknesses of the films built up withPAR10, PAR30, PAR100 or PAR200 as polycations equals 70, 130, 200 or 450nm respectively (FIG. 5). Finally for a given number of depositionsteps, the thickness increases as the molecular weight of PARin-creases.

An opposite behavior was previously demonstrated for multilayer coatingsbuildup with chitosan/HA with various MW of chitosan (Richert 2004,Langmuir). An exponential growth of the coatings was observed for allthe MW of chitosan used, however the coating buildup was more rapid whenthe mass of the chitosan chains was smaller. This behavior was relatedto the diffusive properties of the chitosan chains in the coatings:shorter chains should diffuse more through the coating which should leadto a higher increase in the mass increment after each layer deposition.However in the present study, experimental conditions are different asan homopolypeptide was selected as the polycation instead of apolysaccharide and the range of their chain length was smaller.

Then, coatings with 24 bilayers ((PAR/HA)₂₄) were observed with confocalmicroscopy. In order to label fluorescently the coatings,poly(lysine)-FITC was added as the last layer on top of the coatings.Cross-section images of coatings build up with PAR of different residuenumbers depict thick bands with a green labeling through the wholecoating section (FIG. 2). This indicates that the coatings produce werehomogenously deposited on the surface in all conditions (for PAR with 10to 200 residues).

Next, the antimicrobial properties of (PAR/HA)₂₄ multilayers for PAR ofdifferent number of residues was evaluated. The films were testedagainst a gram positive bacteria, S. aureus, a strain well known to beassociated with nosocomial infections and more particularly withimplant-related infections. For example in the case of orthopaedicimplants, S. aureus with S. epidermis is involved in 70% of infections(biomaterials 84, 2016, 301). S. aureus were incubated for 24 h at 37°C. in the presence of MHB medium on the (PAR/HA)₂₄ coatings. Thebacteria were incubated at high density on surfaces for 24 h at 37° C.in the presence of MHB medium. The normalized growth of pathogens (%)was estimated by comparing absorbance at 620 nm in the presence ofmultilayer films in comparison with the positive control (withoutmultilayer films and in presence of antibiotics in the medium) and thenegative control (without multilayer films and in the absence ofantibiotics in the medium). No significant inhibition was observed forfilms built with PAR10, PAR50, PAR100 and PAR200. However, for PAR30 (30residues), more than 95% of bacterial growth inhibition was observedafter 24 hours. This suggests that PAR30 strongly impact viability of S.aureus. It must be pointed out that the molecular weight effect isextremely striking and up to now such an effect on the multilayer filmfunctionality, whatever this function, was never observed (see FIG. 3).

To evaluate more precisely the health of bacteria in contact with thesurfaces, the respiratory activity of S. aureus using5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was monitored. Healthybacteria will absorb and reduce CTC into an insoluble, red fluorescentformazan product and bacteria which are dead will not reduce CTC andconsequently will not produce fluorescent product (Data not shown). Atotal inhibition of bacteria on (PAR30/HA) surfaces was clearly observedand it was extremely rare to find an area with few bacteria (Data notshown). Comparatively, PAR10, PAR100 or PAR200 surfaces did not preventbacterial adhesion and growth, a similar density of healthy bacteria ason non-treated surfaces was found. This outstanding result is in fullcorrelation with growth inhibition in supernatant described above wherePAR30 was also the only coating strongly effective against bacteria.

In order to elucidate if the bacterial growth inhibition of the(PAR/HA)₂₄ coatings is only limited to S. aureus, bacterial growthinhibition of (PAR30/HA)₂₄ was further tested against other grampositive and gram negative bacteria. Accordingly, the antimicrobialproperties of (PAR30/HA)₂₄ multilayer was evaluated for methicillinresistant S. aureus strain, M. Luteus, E. coli and P. aeruginosa. Theresult shown in FIG. 11 demonstrates that the coating has anantibacterial activity against different gram negative and gram positivebacteria.

In the following the inventors of the present invention wanted tofurther elucidate the underlying mechanism that confer to the coatingsof the invention the strong inhibitory property on the surface and inthe supernatant. As an example the (PAR30/HA)₂₄ coating was furtherinvestigated. Accordingly, the minimal inhibitory concentration (MIC) ofPAR in solution was determined using bacterial assay as described inExperimental Section. For concentrations up to 0.04 mg·mL⁻¹, all PAR(PAR10, PAR30, PAR100 or PAR200) totally inhibited S. aureus growth(FIG. 3). However when PAR concentrations were decreased, a differencebetween the PAR efficiencies was observed: a quasi total inhibition ofS. aureus growth was monitored for all PAR at concentrations of 0.02mg·mL⁻¹ except for the longer one, i.e. PAR200 where only a partialinhibition (about 45%) was measured. Finally for PAR at concentrationsof 0.01 mg·mL⁻¹, inhibition of 100% was shown only for PAR30. Longer orshorter PAR chains (PAR10, PAR100 or PAR200) inhibits only partially(less than 40%) S. aureus growth. This suggests that PAR30 is moreeffective in solution. This reasoning is valid when PAR concentrationvalues are expressed in mass (mg·mL⁻¹) thus it is related to the numberof arginine monomers. However when the graph is plotted withconcentrations in μM (and thus proportional in number of chains), whichmeans that concentration is related to number of chains, differentinterpretation can be made (Data not shown). At low concentrations,longer chains are more effective: at 1 μM, PAR100 and PAR200 totallyinhibit bacterial growth about 90% growth whereas for a similar effectPAR30 and PAR10 needs to be at about 10 μM. Finally, the inventors ofconcluded from these results that all PAR chains are effective insolution to inhibit S. aureus growth at concentrations of 0.2 mg·mL. Fora given mass of PAR chains in the supernatant, PAR30 is the mosteffective. Moreover the MIC₁₀₀ values obtained for PAR30, PAR100 orPAR200 ranges at very low concentrations (between 1 and 2 μM) which isremarkable when compared to well-known antimicrobial peptides (forexample 30 μM for cateslytin with S. aureus, see Adv. Funct. Mater.2013, 23, 4801-4809). PAR is thus a powerful candidate to fight againstS. aureus.

However, PAR chains of different number of residues were not markedlydifferent in their activity in solution and thus the origin of the PAR30activity ob-served with PAR30/HA films is not related to its higheractivity in solution.

To address the conformations of PAR chains and to check if PAR30 chainshave a specific secondary structure that could explain their inhibitoryproperties compare to longer or shorter chains, circular dichroism (CD)experiments were performed. Firstly, secondary structures of PAR chainsin NaCl/TRIS buffer solution (150 mM NaCl, 10 mM Tris, pH 7.4) (Data notshown). All CD spectra of PAR chains (PAR10, PAR30, PAR100 and PAR200)show a unique negative minimum at about 200 nm characteristic of arandom coil conformation in solution. In a second step, PAR conformationin PAR/HA multilayer coatings was monitored (Data not shown).Surprisingly, spectra of the coatings depict totally different profiles:no more minima at 200 nm were observed, however one minima at about 10and another one at about 222 nm were monitored, except for (PAR10/HA)₂₄,which present a unique negative minimum at 200 nm (random coil). Theycan probably not be attributed to HA chains as it is known that insolution at pH 7.4 they have an unordered conformation (Zahouani, ACSApplied Materials, 2016, 8, 14958-14965). Moreover the PAR/HA spectrumcorresponds more probably to chains in α-helix conformationscharacterized by these two minimums. This indicates that PAR chainsshould change from a coil conformation in solution to an α-helix in thecoating. Similar behavior were previously observed for LbL build up withpolylysine and poly(glutamic acid) (Boulmedais F. et al. Langmuir, 2002,18, 4523). Interestingly, unordered antimicrobial peptides are known toadopt an α-helix conformations when they interact with the bacterialmembrane and this mechanism is a key point in their mechanism of action(Porcellini F. et al. 2008, Biochemistry, 47, 5565 and Lugardon K. etal., 2001, J Biol Chem., 276(38): 35875). Here in polyelectrolytemultilayer coatings, PAR chains already adopt an α-helix conformationmost probably due to the interactions between PAR and HA and local highconcentration of PAR. This mechanism can be helpful to fight faster andin a more efficiency way against invading bacteria.

But, because the films built with different PAR chain lengths presentsimilar spectra, the secondary structure of PAR chains cannot explainthe striking molecular weight effect on the bactericidal property of thePAR30/HA films.

In view of the absence of specific properties of PAR30 in solutioncompared to shorter or longer PAR chains, the antimicrobial abilities ofPAR30/HA films should be related to the film property by itself. In thiscontext, the inventors investigated if the bactericidal property of thefilm is due to the release of PAR30 chains from the multilayer into thesolution or if bacteria need to come in contact with the film to bekilled. For this purpose two types of experiments were performed. Usingfluorescently labelled PAR30 chains we first determined the release ofPAR30 chains into the solution containing solely MH medium with andwithout S. aureus. Indeed, a slow release process over a time scale ofthe order of 24 h was observed but it clearly comes out that even after24 h the PAR30 concentration reached in solution lies significantlybelow the corresponding MIC concentration: PAR30 released is about 0.18μM after 24 h while MIC90 is about 2 μM (data not shown). Moreover, whenthe supernatant, after 24 h of contact with the film, was brought incontact with suspension containing bacteria at a final concentrationidentical to previous experiments, absolutely no bacteria growthinhibition was observed, confirming that the MIC was not reached insupernatant (data not shown). Finally, we also performed an experimentwhere bacteria were brought in contact with a (PAR30/HA)₂₄ film for 24hours. Bacteria growth was totally inhibited. The supernatant of thisexperiment was removed and brought it in contact with a fresh suspensionof bacteria. Here again, the bacteria growth was no further inhibited(data not shown). These results demonstrate that the release of thePAR30 chains from the film in the supernatant cannot be at the origin ofthe bactericidal effect. Finally we can hypothesize that thebactericidal effect is directly related to the contact of the bacteriawith the PAR30/HA film which acts as a contact-killing multilayer.

Then the inventors investigated if the bactericidal activity of thePAR30/HA multilayer is related to the mobility of these chains in thefilms. Indeed, it is known that the exponential character of amultilayer is related to the diffusion ability of at least one of itsconstituents in and out of the film during each deposition step. Theyfirst determined the mobility of the different chains, PAR10, PAR30,PAR100 and PAR200 in the (PAR/HA)₂₄ multilayer by using FRAP method.FIG. 7 shows the evolution of the normalized fluorescence in thebleached area as a function of the square root of time. Recovery offluorescence appears very fast for PAR10 and PAR30 compare to PAR100 orPAR200. From these curves we can deduce the percentage of mobile chainsover the timescale of the experiments. It clearly appears that PAR10 andPAR30 chains are more mobile (between 85 to 90% of mobile fraction) thanthe PAR100 (only 63% of mobile fraction). The largest chains PAR200correspond to the slowest with about 12% of the population which ismobile (FIG. 8). Accordingly, the fraction of mobile chains dramaticallydecreases when the chain length increases from 30 to 100 or 200residues.

To confirm the dependence of mobility on the bactericidal effect of thefilm, the inventors cross-linked the PAR30/HA multilayers using astandard EDC-NHS cross-linking method which creates a covalent linkbetween amine groups of PAR and carboxylic groups of HA. The proportionof mobile chains measured by FRAP method decreases significantly from88% for the non-crosslinked film to 20% for the cross-linked one (Datanot shown). When the cross-linked film was brought in contact with S.aureus, only about 40% of inhibition of the bacterial growth wasobserved after 24 h of contact (FIGS. 9 and 10).

These results suggest that the percentage of mobile PAR chains in themultilayer is an important parameter controlling its bactericidalproperty of the film (FIG. 8).

Finally, using confocal microscopy, the inventors also investigated thestructure of the film after 24 hours of contact with bacteria (Data notshown). For this purpose, films were constructed by incorporatingfluorescently labelled PAR30 chains. It was found that after 24 hours ofcontact, the film is no longer homogeneous but that non-fluorescentareas appear. Because these areas have a smooth shape, they suggest areorganization of the film. Such a behavior is not observed in theabsence of bacteria where the films remain homogeneous. Such areorganization could be consecutive to de decrease of the PAR chains inthe film and suggest the following bactericidal mechanism: When bacteriacome in contact with the multilayer, the negative bacteria membranes actas strong attractive surfaces for the PAR chains. Mobile PAR chains arethus soaked out of the film by the bacteria membranes and destabilizethem. Chains of large molecular weight have a stronger membranedestabilization power than smaller molecular weight ones as it comes outfrom the MICs determined in solution. PAR10 chains are 10 times lessactive than PAR100 or PAR200 chains but PAR30 chains are only 2 timesless active than PAR100 or PAR200 chains. Yet, in a film one can assumethat the concentration of arginine monomers is fairly independent of themolecular weight of the PAR chains. Thus, the concentration of chainsdecreases when the molecular weight of the polyelectrolytes increases.For example the concentration PAR30 chains in the film should be 3 timeshigher than that of PAR100. In addition there are of the order of 90% ofPAR10 chains that are mobile whereas only 70% of PAR100 chains aremobile. This leads to 4 times more PAR10 chains than PAR100 in the film.There could be other factors explaining the higher propensity of filmsPAR/HA films built with PAR30 to be strongly bactericidal but the chainmobility is without doubt an important one.

The inventors clearly demonstrate that the property of the coating isrelated to the length of the PAR polyelectrolyte chain. Theconcentration of the mobile PAR chains is a key-factor in theantimicrobial effectiveness and thus the films having 24 layers andbeing buildup with PAR containing 30 residues of arginine seem optimalfor such bioactivity.

PAR containing 10 residues per chain seems not active in (PAR/HA)₂₄films despite its high mobility in the films and its MIC which is closeto that of PAR30. The inventors considered that this can be attributedto the film buildup which is about two times thinner with PAR10 comparedto PAR30. After 24 bilayers, the amount of free PAR10 chains able toinhibit for 24 h bacterial growth could be too low.

To verify this hypothesis, the inventors performed therefore additionalexperiments with PAR10/HA films containing a higher number of bilayer(48 instead of 24) (FIG. 13). PAR10/HA films containing 48 bilayersbecome antimicrobial, however PAR100/HA or PAR200/HA remain inactivewith 48 bilayers.

This indicates that a sufficient number of free PAR chains should beavailable to confer antimicrobial properties to the films. For PAR10 orPAR30, this number is reached with 48 or 24 bi-layers respectively.

The inventors consider that, concerning the mechanism of action of PAR30(or PAR10 for thicker films), it should be related to diffusion of PAR30or PAR10 chains out of the film enhanced by the attractive electrostaticinteractions between the positively charged PAR and the negativelycharged bacterial membrane. This interaction should occur as soon as thebacteria come in contact with the PAR/HA film. Time-lapse microscopyexperiments have clearly shown that the bacteria are killed when theytouch the PAR30/HA surface (data not shown). As PAR chains are mobile,they can diffuse and stick to the membrane. Then the mechanism should beclosed to the mechanism of action of antimicrobial peptides, which arepositively charged peptides that interact with bacterial membrane.

In order to investigate the biocompatibility of the PAR/HA coatings, theinventors seeded human primary fibroblasts from skin with medium thatwas in contact for 24 h with (PAR30/HA)₂₄ glass slides. After 24 h ofseeding, no sign of toxicity was observed, the viability was equivalentto control conditions, i.e. glass surfaces (data not shown). Thispreliminary test demonstrates that the PAR released in the presence ofmedium in the supernatant shows no apparent sign of toxicity for theprimary cells used. This is a positive point in the perspective of theapplication of PAR/HA films as coatings of implanted medical devices.

The inventors further investigated the biocidal activity of the coatingsof the invention, in particular of (PAR30/HA)₂₄ coatings, over more than24 hours. Therefore, a (PAR30/HA)₂₄ multilayer coating was put incontact with a fresh suspension of S. aureus after each 24 h and the(PAR30/HA)₂₄ shows after 24 and 48 h of incubation a total inhibition ofbacteria and a bacterial growth that is strongly reduced (by 70%) after72 h. This demonstrates the biocidal activity of the coatings of theinvention over 3 days and three successive contaminations (FIG. 18).

To summarize, the inventors of the present invention have found thatmultilayer coatings comprising PAR, in particular PAR30 and PAR10, aspolycation and HA as polyanion present a strong anti-microbial effectagainst S. aureus, M. Luteus, E. coli and P. aeruginosa. The inventorsdemonstrated in context of PAR30 coatings that this effect strikinglydepends on the molecular weight of the polypeptide chains. This effectis explained by the concentration of mobile in the multilayers and theirpower to kill bacteria as a function of the molecular weight. Thismechanism can be transferred to PAR10, because PAR30 as well as PAR30both demonstrate high mobility.

However, under the conditions tested PAR10 films require more than 24bilayers in order to be active. As demonstrated by the inventors sincePAR10 and PAR30 have several capabilities in common the activity seemsto depend on the amount of PAR10 or PAR30 present in the films and PAR10biofilms require more PAR10 layers to obtain the same biocidal activityas PAR30 films.

These results open the route to new type of applications ofpolyelectrolyte multilayers, in particular of antibacterial multilayers,where the function can be tuned by the molecular weight of thepolyelectrolytes.

1. A polyelectrolyte coating, comprising: (a) at least one polycationiclayer consisting of at least one polyarginine consisting of n repetitivearginine units, wherein n is an integer comprised between 2 and 10, and(b) at least one polyanionic layer consisting of hyaluronic acid.
 2. Thepolyelectrolyte coating according to claim 1, which comprises 25 to 500polycationic layers, preferably 30 to 100 polycationic layers, morepreferably 40 to 80 polycationic layers.
 3. The polyelectrolyte coatingaccording to claim 1, which comprises 25 to 500 polyanionic layers,preferably 30 to 100 polyanionic layers, more preferably 40 to 80polyanionic layers.
 4. The polyelectrolyte coating according to claim 1,wherein the number of the polycationic layers and the number of thepolyanionic layers are the same or differ by one layer.
 5. Thepolyelectrolyte coating according to claim 1, wherein said coating hasbiocidal activity.
 6. The polyelectrolyte coating according to claim 1,wherein said coating further comprises a pharmaceutical active drug. 7.The polyelectrolyte coating according to claim 1, wherein said coatingis biocompatible.
 8. A device comprising a polyelectrolyte coating,wherein said polyelectrolyte coating comprises: (a) at least onepolycationic layer consisting of at least one polyarginine consisting ofn repetitive arginine units, wherein n is an integer comprised between 2and 10, and (b) at least one polyanionic layer consisting of hyaluronicacid.
 9. The device of claim 8, wherein the polyelectrolyte coatingcovers at least a portion of the surface of said device.
 10. The deviceof claim 8, wherein said device is a medical device.
 11. The device ofclaim 10, wherein said device is an implantable device.
 12. Theimplantable device according to claim 11, wherein the implantable deviceis selected from the group comprising catheters, arteriovenous shunts,breast implants, cardiac and other monitors, cochlear implants,defibrillators, dental implants, maxillofacial implants, middle earimplants, neurostimulators, orthopedic devices, pacemaker and leads,penile implants, prosthetic devices, replacement joints, spinalimplants, voice prosthesis, artificial hearts, contact lenses, fracturefixation device, infusion pumps, intracranial pressure device,intraocular lenses, intrauterine devices, joint prosthesis, mechanicalheart valves, orthopedic devices, suture materials, urinary stents,vascular assist device, vascular grafts, vascular shunts and vascularstents, and artificial vessels of permanent or transient types.
 13. Amethod for preparing a device comprising a polyelectrolyte coating, themethod comprising: (a) providing a device; (b1) depositing on thesurface of said device (i) at least one polycationic layer consisting ofat least one polyarginine consisting of n repetitive arginine units,wherein n is an integer comprised between 2 and 10, and then ii) atleast one polyanionic layer consisting of hyaluronic acid, or (b2)depositing on the surface of said device ii) and then i) as definedabove, and optionally repeating step b1) and/or b2).
 14. The method forpreparing a device according to claim 13, further comprising at leastone washing step after step i) and/or ii) of b1) or b2).
 15. The methodfor preparing a device according to claim 14, further comprising atleast one drying step after step i) and/or ii) of b1) or b2) and/or theat least one washing step.
 16. A kit comprising a) at least onepolycationic material consisting of at least one polyarginine consistingof n repetitive arginine units, wherein n is an integer comprisedbetween 2 and 10, and b) at least one polyanionic material consisting ofhyaluronic acid.
 17. A method of preventing a bacterial infection in anindividual undergoing an implantation of an implantable devicecomprising the steps of: i) providing an implantable device comprising apolyelectrolyte coating, wherein said polyelectrolyte coating comprises:(a) at least one polycationic layer consisting of at least onepolyarginine consisting of n repetitive arginine units, wherein n is aninteger comprised between 2 and 10, and (b) at least one polyanioniclayer consisting of hyaluronic acid, and ii) implanting said implantabledevice in the individual, wherein said implantable device prevents abacterial infection.
 18. The method of claim 17, wherein the bacterialinfection is a nosocomial infection.